Endocytic recycling is essential for the control of receptors on the plasma membrane in mammalian cells. Consequently, recycling impacts health by regulating crucial cellular events such as signal transduction and proliferation, cholesterol homeostasis, nutrient uptake, and insulin-dependent glucose transport. Thus, understanding the regulation of endocytic events is critical for a wide range of diseases, including malignancies, heart disease and diabetes. Endocytic transport and recycling are controlled by a number of small Rab GTP-binding proteins. Recently, a non-Rab protein called EHD1 has been ascribed a role in regulating recycling at the recycling compartment. However, the mode by which EHD1 coordinates its regulatory activity with Rab-family proteins is not understood. A biochemical approach has identified the divalent Rab4/Rab5 effector prtoein, Rabenosyn-5, as a binding partner for EHD1, and defined a role for it in recycling at the early endosome. The critical task at hand is to understand how trafficking events at the early endosome are linked to those at the recycling compartment, particularly what regulates transport and fusion of early endosome-derived vesicles with the recycling compartment. The first aim will focus on identifying the mechanisms by which proteins are transported from early endosomes to the endocytic recycling compartment, en route to the plasma membrane. The working hypothesis is that the interaction between EHD1 and Rabenosyn-5 is critical for transport of internalized proteins from early endosomes to the recycling compartment. The second aim is based on new data elucidating a physical connection between EHD proteins and a Rab11 effector protein, and proposes to determine the mechanisms by which EHD proteins coordinate endocytic recycling and transport with Rab11 and SNARE proteins. The working hypothesis is that EHD proteins coordinate transport steps with Rab11 and its effectors, and that the SNARE proteins Syntaxin13 and SNAP29 play a critical role in fusion of early endosome-derived vesicles at the recycling compartment. These aims will be accomplished using novel fibroblasts from EHD1-knock-out mice, RNAi- based 'knock-down/knock-in' strategy, and a series of biochemical, flow cytometry and microscopic assays. These studies will significantly enhance our fundamental understanding of the mechanisms regulating recycling and have an important bearing on diseases as diverse as cancer and atherosclerosis and diabetes.